Project Summary/Abstract Natural products including polyketides, fatty acids, amino acids, terpenoids and steroids, are playing an increasingly important role in the pharmaceutical industry. Though many of the natural products are discovered in plants, mass production of these molecules relies on culturing microorganisms in bioreactors. However, it is often a challenge to develop and maintain high biomanufacturing productivity and yield to enable low-cost and high-quality production at large scales. One unmet need is an ability to rapidly and accurately measure the physiological status of microbes at the cellular level within bioreactors. Measuring the cellular metabolic state, especially energetic and redox parameters is key to guiding decisions on feeding strategies and other operation actions. Due to the complex nature of bioreactor cell culture systems, direct measurements of cellular metabolic parameters is current impractical, and relies on periodic removal of culture samples for off-line analysis. However, frequent sampling introduces time delays, contamination risks, and increased manufacturing costs. During this R&D program, Physical Sciences Inc. (PSI) in collaboration with the University of Massachusetts Lowell (UML) will develop a novel two-photon excitation (TPE) fluorescence redox sensor for on-line, real-time measurement of cell metabolism in bioreactors for natural products fermentation. A robust TPE fiber probe will be developed that can be sterilized and inserted into bioreactor cultures for continuous measurement of dynamic changes of important intracellular metabolites. The intracellular redox ratio will be monitored using the autofluorescence of endogenous fluorophores, eliminating the need for exogenous fluorescence labeling. Development of this versatile, fiber-based redox probe draws on innovative techniques including high-throughput delivery of femtosecond laser pulses through a specialty fiber cord and efficient signal collection using a unique fiber bundle design. During this proposed Phase I program, the PSI led team will fabricate a prototype redox sensor and evaluate its performance in yeast bioreactors. Proof of concept experiments will be performed to demonstrate the effectiveness of the prototype for on-line, continuous metabolic measurement of microorganisms in natural products fermentation bioreactors. The real-time redox based metabolic measurement will be compared with standard off-line metabolism measurements. The correlation between cell ratiometric redox assessment and the bioprocessing yield will be investigated, demonstrating the value of this sensing technology in guiding effective operation actions for improved productivity during bioprocessing of natural products. During a subsequent Phase II program the PSI team will develop the technology to maturity and conduct large-scale biological and process development studies to demonstrate its broad application in biomanufacturing processes.